Nitric Oxide Inhibits Blue Light-Specific Stomatal Opening Via Abscisic Acid Signaling Pathways in Vicia Guard Cells

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1 Plant Cell Physiol. 48(5): (2007) doi: /pcp/pcm039, available online at ß The Author Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please Nitric Oxide Inhibits Blue Light-Specific Stomatal Opening Via Abscisic Acid Signaling Pathways in Vicia Guard Cells Xiao Zhang 1, 2, Atsushi Takemiya 1, Toshinori Kinoshita 1 and Ken-ichiro Shimazaki 1, * 1 Department of Biology, Faculty of Science, Kyushu University, Ropponmatsu, Fukuoka, Japan 2 Henan Key Laboratory of Plant Stress Biology, College of Life Sciences, Henan University, Kaifeng , PR China Recent evidence suggests that nitric oxide (NO) acts as an intermediate of ABA signal transduction for stomatal closure. However, NO s effect on stomatal opening is poorly understood even though both opening and closing activities determine stomatal aperture. Here we show that NO inhibits stomatal opening specific to blue light, thereby stimulating stomatal closure. NO inhibited blue light-specific stomatal opening but not red light-induced opening. NO inhibited both blue light-induced H þ pumping and H þ -ATPase phosphorylation. The NO scavenger 2-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-ptio) restored all these inhibitory effects. ABA and hydrogen peroxide (H 2 O 2 ) inhibited all of these blue light-specific responses in a manner similar to NO. c-ptio partially restored the ABAinduced inhibition of all of these opening responses but did not restore inhibition of the responses by H 2 O 2. ABA, H 2 O 2 and NO had slight inhibitory effects on the phosphorylation of phototropins, which are blue light receptors in guard cells. NO inhibited neither fusicoccin-induced H þ pumping in guard cells nor H þ transport by H þ -ATPase in the isolated membranes. From these results, we conclude that both NO and H 2 O 2 inhibit blue light-induced activation of H þ -ATPase by inhibiting the component(s) between phototropins and H þ -ATPase in guard cells and stimulate stomatal closure by ABA. Keywords: Abscisic acid Blue light H þ pumping Nitric oxide Stomatal opening. Abbreviations: c-ptio, 2-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; NO, nitric oxide; SNP, sodium nitroprusside; Vfphot, Vicia faba phototropin. Introduction Nitric oxide (NO) is a highly diffusible gas and a ubiquitous bioactive molecule with well-characterized signaling roles in mammalian systems (Furchgott 1995). NO is suggested to play crucial roles in plant development, stress responses and programmed cell death, but its site of action in any signaling pathway remains unknown (Delledonne et al. 1998, Garcia-Mata and Lamattina 2003, Garcia-Mata et al. 2003, Gould et al. 2003). Recently, NO was implicated in stomatal closure in response to ABA (Garcia-Mata and Lamattina 2002, Neill et al. 2002a). NO stimulates the elevation of cytosolic Ca 2þ by the release of intracellular stores in guard cells, thereby inhibiting the inward-rectifying K þ channels (I K,in ) and activating anion channels (I cl ). These changes in the channel activities cause stomatal closure as Ca 2þ -dependent pathways in ABA signaling (Garcia-Mata et al. 2003). Such NO-dependent Ca 2þ release in guard cells is regulated by protein phosphorylation/dephosphorylation, because general serine/threonine protein kinase inhibitors simultaneously suppressed both the inactivation of I K,in and the activation of I cl by NO (Sokolovski et al. 2005). Furthermore, NO has been demonstrated to cause stomatal closure in the epidermal peels of Vicia and Arabidopsis, and ABA-induced NO synthesis has been found to require hydrogen peroxide (H 2 O 2 ) production, with NO synthesis induced mainly by nitrate reductase in guard cells (Desikan et al. 2002, L8 et al. 2005, Bright et al. 2006). These investigations focused on NO s effect on the processes of stomatal closure, and suggested that NO enhances plant tolerance to drought stress (Garcia-Mata and Lamattina 2001, Garcia-Mata and Lamattina 2002, Neill et al. 2002a, Neill et al. 2002b). However, little is known about NO s effect on stomatal opening by light, although ABA inhibits the stomata s opening responses (Shimazaki et al. 1986, Goh et al. 1996, Assmann and Shimazaki 1999). Stomatal opening to light consists of two distinct components: one depends on photosynthesis as an energy source induced by a relatively strong light, while the other depends on the signaling system specific to a weak blue light (Shimazaki et al. 2007). Photosynthesis-dependent stomatal opening is mediated by the decrease in intercellular CO 2 concentration by mesophyll chloroplasts and/or guard cell chloroplast activity, although the exact nature of these processes is unknown (Roelfsema and Hedrich 2005, Vavasseur and Raghavendra 2005). Separate lines of evidence have demonstrated that blue light activates the H þ pump in guard cells (Assmann et al. 1985, Shimazaki et al. 1986, Roelfsema et al. 2001, Taylor and Assmann 2001). The activated pump increases the insidenegative electrical potential across the plasma membrane, *Corresponding author: , kenrcb@mbox.nc.kyushu-u.ac.jp; Fax, þ

2 716 Inhibition of BL-specific stomatal opening by NO drives K þ accumulation through the voltage-gated inwardrectifying K þ channels and finally results in stomatal opening (Assmann and Shimazaki 1999, Schroeder et al. 2001, Roelfsema and Hedrich 2005). Recent studies have demonstrated that the H þ pump is the plasma membrane H þ -ATPase and that its activation by blue light is mediated via the phosphorylation of threonine residues in the C-terminus with a subsequent binding of a protein (Kinoshita and Shimazaki 1999, Kinoshita and Shimazaki 2002). Phototropins (phot1 and phot2) have been identified as plant-specific blue light receptors in the phototropism of Arabidopsis thaliana (Huala et al. 1997, Briggs et al. 2001) and also mediate blue light responses of plants, including phototropism, chloroplast movement, leaf expansion, rapid inhibition of hypocotyl elongation, leaf movement and stomatal opening (Kagawa et al. 2001, Kinoshita et al. 2001, Sakai et al. 2001, Briggs and Christie 2002, Sakamoto and Briggs 2002, Folta et al. 2003, Inoue et al. 2005). Phototropins are serine/threonine protein kinases with two LOV (light, oxygen and voltage) domains (Christie et al. 1998, Salomon et al. 2000), and the activated phototropins undergo autophosphorylation with a subsequent binding of proteins (Kinoshita et al. 2003), and ultimately activate the plasma membrane H þ -ATPase via signal cascades in guard cells. However, knowledge about blue light signaling pathways from phototropins to H þ -ATPase in guard cells is largely limited, although several components, including cytosolic Ca 2þ, PRT2 (ROOT PHOTOTROPISM2), type 1 protein phosphatase and protein kinase in the plasma membrane have been suggested to play key roles (Kinoshita et al. 1995, Shimazaki et al. 1999, Svennelid et al. 1999, Inada et al. 2004, Takemiya et al. 2006). In this study, we investigated NO s effect on lightinduced stomatal opening in Vicia faba guard cells to clarify the involvement of NO in ABA signaling. We found that NO inhibits stomatal opening by suppressing blue lightdependent phosphorylation of the plasma membrane H þ -ATPase, and that the inhibition by NO occurs on the component(s) between phototropins and the plasma membrane H þ -ATPase. Results NO inhibits blue light-specific stomatal opening We investigated NO s effects on light-induced stomatal opening in the epidermis of V. faba. Stomata opened slightly by relatively strong red light at 150 mmol m 2 s 1 and greatly by blue light at 15 mmol m 2 s 1 superimposed on the red light. Sodium nitroprusside (SNP), an NO donor, had no effect on either stomatal aperture in the dark or opening response by red light. However, SNP completely inhibited blue lightspecific stomatal opening (Fig. 1). The NO scavenger Fig. 1 Inhibition of blue light (BL)-dependent stomatal opening in epidermal peels by nitric oxide (NO). Epidermal peels were preincubated for 2 h in the dark (Dark), and then illuminated with red light at 150 mmol m 2 s 1 (RL) or superimposed with blue light at 15 mmol m 2 s 1 on the red light (RLþBL) in the presence of sodium nitroprusside, an NO donor (SNP, 100 mm) or the specific NO scavenger 2-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1- oxyl-3-oxide(c-ptio, 100 mm) for another 2 h. Each bar indicates a mean of measurements with standard errors from three independent experiments. An asterisk shows significant differences with a P (t-test) vs. SNP treatment alone. 2-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3- oxide (c-ptio), which was previously shown to block NO s effects (Delledonne et al. 1998, Neill et al. 2002a), largely restored this inhibitory effect. c-ptio alone did not alter stomatal aperture in any of the light conditions. These results indicate that NO inhibits blue light-specific stomatal opening in Vicia epidermal peels. NO inhibits blue light-dependent H þ pumping Since blue light-dependent H þ pumping acts as a driving force for stomatal opening (Assmann et al. 1985, Shimazaki et al. 1986), the NO-dependent inhibition of stomatal opening prompted us to investigate NO s effects on H þ pumping. Blue light-dependent H þ pumping in Vicia guard cell protoplasts was inhibited by about 80% after application of SNP at 100 mm (Fig. 2A), and this inhibition was dependent on both the incubation time and the SNP concentration (Fig. 2B). Since blue light-dependent H þ pumping is mediated by the plasma membrane H þ -ATPase via phosphorylation of the C-terminus with a subsequent binding of a protein (Kinoshita and Shimazaki 1999, Zhang et al. 2004), the levels of H þ -ATPase phosphorylation were determined through the amount of bound proteins. As shown in Fig. 2C, protein blot analysis indicated that SNP inhibited the binding of a protein to the H þ -ATPase in a concentrationdependent manner. c-ptio almost completely prevented

3 Inhibition of BL-specific stomatal opening by NO 717 Fig. 3 Restoration of NO-induced inhibition of blue light (BL)-dependent H þ pumping and the binding of a protein to the H þ -ATPase by c-ptio. (A) Effects of c-ptio on blue lightdependent H þ pumping in the presence of SNP. Guard cell protoplasts were pre-incubated under background red light (RL) for 40 min and then illuminated with a pulse of BL. c-ptio at 100 mm and SNP at 100 mm were added to guard cell protoplast suspension 40 and 30 min before the BL pulse, respectively. Values are the means of three independent experiments with standard errors. (B) Effects of c-ptio on the levels of a protein binding to the H þ -ATPase in the presence of SNP determined by protein blot analysis. Immunoblot analysis of the H þ -ATPase. Other reaction conditions were the same as shown in (A). Arrowheads indicate the H þ -ATPase. Fig. 2 Inhibition of blue light (BL)-dependent H þ pumping and the binding of a protein to the H þ -ATPase in guard cell protoplasts from Vicia faba by NO. (A) Time-dependent SNP inhibition of BL-dependent H þ pumping. Guard cell protoplasts were pre-incubated under background red light (RL, 600 mmol m 2 s 1 ) for 40 min and then illuminated with a BL pulse (100 mmol m 2 s 1, 30 s). SNP (100 mm) was added to guard cell protoplast suspension at the indicated times before the application of the BL pulse. Values are the means of three independent experiments with standard errors. (B) Dose-dependent inhibition of BL-dependent H þ pumping by SNP. SNP was added to guard cell protoplast suspension at the indicated concentrations 30 min before the application of the BL pulse. Values are the means of three independent experiments with standard errors. Experimental conditions are the same as shown in (A). (C) Protein blot analysis of H þ -ATPase with and without SNP. Recombinant glutathione S-transferase (GST) protein was used as a probe. Immunoblot analysis of H þ -ATPase with and without SNP. Rabbit antisera to the plasma membrane H þ -ATPase were used. Guard cell protoplasts were completely solubilized 2.5 min after the BL pulse and subjected to SDS PAGE, and then electroblotted onto nitrocellulose membranes. Other reaction conditions are the same as shown in (B). Arrowheads indicate the position of the H þ -ATPase. SNP-mediated inhibition in both blue light-dependent H þ pumping and H þ -ATPase phosphorylation (Fig. 3A, B). c-ptio alone affected neither blue light-dependent H þ pumping nor H þ -ATPase phosphorylation levels. These results verified that NO inhibited blue light-dependent H þ pumping by suppressing the phosphorylation of the plasma membrane H þ -ATPase. NO s effect on fusicoccin-dependent H þ pumping The plasma membrane H þ -ATPase is the terminal target of blue light signaling in guard cells (Kinoshita and Shimazaki 1999). NO s inhibition of protein kinase in blue light signaling pathways may explain why NO inhibits plasma membrane H þ -ATPase phosphorylation. To solve this issue, a fungal toxin fusicoccin, which functions as a direct stimulator of H þ -ATPase phophorylation by bypassing blue light signaling pathways (Shimazaki et al. 1992, Kinoshita and Shimazaki 2001, Zhang et al. 2004),

4 718 Inhibition of BL-specific stomatal opening by NO Fig. 4 Effects of NO on both fusicoccin (FC)-dependent H þ pumping and the binding of a protein to the H þ -ATPase in guard cell protoplasts. (A) SNP was added at the indicated concentrations 50 min before the application of FC. Values are the means of three independent experiments with standard errors. (B) Guard cell protoplasts were completely solubilized 5 min after FC application, and solubilized guard cell protoplast proteins were separated by SDS PAGE. Binding of a protein to H þ -ATPase was determined by protein blot analysis. Immunoblot analysis of H þ -ATPase in the presence of SNP. Other experimental conditions were the same as shown in (A). Arrowheads indicate H þ -ATPase. was used. Fusicoccin induced both activation of H þ pumping and phosphorylation of the H þ -ATPase in Vicia guard cell protoplasts. SNP had little inhibitory effect on either fusicoccin-induced H þ pumping (Fig. 4A) or binding of a protein to the H þ -ATPase (Fig. 4B). We isolated the microsomal fractions from guard cells and determined NO s effect on the H þ pumping mediated by the H þ -ATPase in these vesicles. SNP had no effect on the H þ pumping (data not shown). These data indicated that NO inhibited neither the protein kinase nor the H þ -ATPase activity and might inhibit some signal component(s) upstream of the protein kinase in blue light signaling of guard cells. Effects of NO, ABA and H 2 O 2 on the phosphorylation of phototropins and H þ -ATPase In stomatal opening, phototropins have been demonstrated to function as blue light receptors and to autophosphorylate in response to blue light, and the phosphorylation activity is required for the signaling (Briggs and Christie 2002, Kinoshita et al. 2003). We suspected that NO might act on phototropin activities because NO affects cysteine sulfhydryl groups in the protein by nitrosylation, and phototropins require the sulfhydryl groups for their photochemical reactions in LOV domains (Salomon et al. 2000, Sokolovski and Blatt 2004). However, autoradiograms revealed that SNP at 100 mm slightly (by 520%) inhibited the blue light-dependent autophosphorylation of V. faba phototropins (Vfphots) (Fig. 5A), but strongly (by 70%) inhibited blue light-induced phosphorylation of H þ -ATPase (Fig. 5B). The mobility shift of Vfphots to the upper side by blue light strengthened the occurrence of autophosphorylation in the presence of SNP. There was no change in the amounts of Vfphots by these treatments. These results indicated that NO mainly inhibited the component(s) downstream of the phototropins and upstream of the H þ -ATPase in the blue light signaling pathway of guard cells. In view of the conclusion that NO functions downstream of H 2 O 2 in ABA signaling for stomatal closure (L8 et al. 2005, Bright et al. 2006), the results shown above prompted us to investigate whether or not ABA and H 2 O 2 have similar effects on Vfphots and the H þ -ATPase. Like NO, ABA and H 2 O 2 slightly (by 10 and 14%, respectively) inhibited autophosphorylation of Vphots (Fig. 6A), but drastically (by 69 and 73%, respectively) inhibited phosphorylation of the H þ -ATPase (Fig. 6B). The inhibitory actions of both ABA and H 2 O 2 on the blue light responses of guard cells were essentially the same as that of NO. These results, together with those of previous works (Pei et al. 2000, Zhang et al. 2001, Desikan et al. 2002, Garcia-Mata and Lamattina, 2002, Zhang et al. 2004, L8 et al. 2005, Bright et al. 2006), suggest that NO probably acts downstream of H 2 O 2 as an intermediate of ABA signaling pathways in guard cells. c-ptio partially restores ABA inhibition of responses but not H 2 O 2 inhibition of responses Finally, we tested whether or not c-ptio also restores inhibition of stomatal opening, of H þ pumping, and of H þ -ATPase phosphorylation by ABA or H 2 O 2. c-ptio partially restored the ABA-induced inhibition of all three, but had no effect on H 2 O 2 -induced inhibition (Fig. 7A C). These results most plausibly indicate that ABA induces the production of H 2 O 2 and NO, and that both H 2 O 2 and NO inhibit H þ -ATPase phosphorylation (Zhang et al. 2004, Bright et al. 2006). We determined the amounts of H þ -ATPase in all of these experiments and showed that none of the treatments had any effect on the amounts (Figs. 2C, 3B, 4B, 5B, 6B, 7C).

5 Inhibition of BL-specific stomatal opening by NO 719 Fig. 5 Effects of NO on the levels of phosphorylation of phototropins (Vfphots) and the plasma membrane H þ -ATPase in guard cell protoplasts from Vicia faba. Guard cell protoplasts were pre-incubated with [ 32 P]orthophosphate for 60 min under background red light and were exposed to 100 mm SNP for 30 min before the application of the BL pulse. (A) Autoradiogram of immunoprecipitated Vfphots. The reaction was terminated by adding an equal volume of solubilizing medium to the guard cell protoplast suspension 1 min after the BL pulse. In each lane, immunoprecipitated Vfphots was obtained from 200 mg of guard cell proteins. Immunoblot analysis of Vfphots. The reaction was terminated by adding trichloroacetic acid to the final concentration of 10% (w/v) to the guard cell protoplast suspension (70 mg) 1 min after the BL pulse. (B) Autoradiogram of immunoprecipitated H þ -ATPase. The reaction was terminated by adding an equal volume of solubilizing medium to the guard cell protoplast suspension 2.5 min after the BL pulse. In each lane, the immunoprecipitated H þ -ATPase was obtained from 100 mg of guard cell proteins. Immunoblot analysis of H þ -ATPase. In each lane, the immunoprecipitated H þ -ATPase was obtained from 50 mg of guard cell proteins using anti-h þ -ATPase antibody conjugated to protein A agarose with dimethyl pimelimidate dihydrochloride (DMP). Arrowheads indicate the Vfphots or H þ -ATPase. The asterisk indicates a non-specific protein. Discussion Specific inhibition of stomatal blue light response ABA is known to cause the production of NO and H 2 O 2, and closes stomata to prevent water loss during drought. However, NO s effect on stomatal opening processes has not been investigated yet. In this study, we found that NO inhibits stomatal opening, which is specific to blue light, but does not inhibit red light-induced stomatal opening using SNP as an NO generator (Fig. 1). Since blue light-induced stomatal opening is driven by H þ pumping Fig. 6 Effects of ABA and H 2 O 2 on the levels of phosphorylation of Vfphots and the plasma membrane H þ -ATPase in guard cell protoplasts from Vicia faba. Guard cell protoplasts were preincubated under background red light for 60 min and then illuminated with a pulse of BL. ABA at 10 mm orh 2 O 2 at 1 mm were added 30 min before the application of the BL pulse. Other experimental conditions were the same as shown in Fig. 5. across the plasma membrane through the H þ -ATPase activities in guard cells, we investigated whether or not NO affects the pump activities. We found that blue lightdependent H þ pumping was inhibited by NO in both a time- and concentration-dependent manner (Fig. 2) and that phosphorylation of the H þ -ATPase was inhibited in a similar manner. These results indicate that inhibition of blue light-dependent H þ -ATPase phosphorylation is responsible for NO s inhibition of blue light-specific stomatal opening. Site of NO inhibition in blue light signaling pathways As for the site of NO inhibition, several candidates are present in the blue light signaling pathways of guard cells. We excluded the direct inhibition of the H þ -ATPase because NO did not inhibit the H þ transport in the isolated membranes. In the plasma membrane of guard cells, there is an unidentified protein kinase that may directly phosphorylate H þ -ATPase by stimulation with a fungal toxin, fusicoccin. The kinase activity is actually insensitive to general protein kinase inhibitors (Kinoshita and Shimazaki 2001), and its activity that phosphorylates H þ -ATPase has been demonstrated in the plasma membrane of spinach leaves (Svennelid et al. 1999). It is possible that NO inactivates this protein kinase activity, thereby suppressing H þ -ATPase phosphorylation. We tested NO s effect on this process. However, NO did not inhibit fusicoccin-induced phosphorylation of H þ -ATPase, suggesting that NO did not affect this protein kinase. These results suggest the site of NO inhibition should be upstream of the protein kinase, which may phosphorylate the H þ -ATPase in guard cells. Since H þ -ATPase received the signal for its activation

6 720 Inhibition of BL-specific stomatal opening by NO Fig. 7 Effects of c-ptio on the inhibition of stomatal opening, of BL-dependent H þ pumping, and of the protein binding level by ABA or H 2 O 2. (A) Effects of ABA (10 mm) and H 2 O 2 (1 mm) on BL-dependent stomatal opening in the presence or absence of c-ptio (100 mm). Other experimental conditions were the same as shown in Fig. 1. Each bar indicates a mean of measurements with standard errors from three independent experiments. An asterisk shows significant differences with a P (t-test) vs. ABA treatment alone. (B) Effects of c-ptio on BL-dependent H þ pumping in the presence of ABA or H 2 O 2. c-ptio at 100 mm and ABA at 10 mm orh 2 O 2 at 1 mm were added to guard cell protoplast suspension 40 and 30 min before the BL pulse, respectively. Values are the means of three independent experiments with standard errors. Other experimental conditions were the same as shown in Fig. 3a. (C) Effects of c-ptio on the levels of a protein binding to the H þ -ATPase in the presence of ABA or H 2 O 2 determined by protein blot analysis. Guard cell protoplasts were completely solubilized 2.5 min after the BL pulse. Solubilized guard cell protoplast proteins were separated by SDS PAGE. Arrowheads indicate the H þ -ATPase. Other reaction conditions were the same as shown in (B). through phototropins, which are blue light receptor kinases, phototropins can be a site of NO inhibition. We therefore determined NO s effects on the autophosphorylation of Vfphots and on H þ -ATPase phosphorylation in response to blue light in the same preparations. The results indicate that NO slightly inhibited the phosphorylation of Vfphots and greatly inhibited that of H þ -ATPase. These results, together with the observations above, suggest that NO mainly inhibits the signal component(s) between phototropins and H þ -ATPase. We have recently demonstrated that type 1 protein phosphatase mediates the signaling between phototropins and the plasma membrane H þ -ATPase in Vicia guard cells (Takemiya et al. 2006). We tested NO s effect on the catalytic activity of recombinant type 1 protein phosphatase expressed in Escherichia coli and found that NO did not affect the activity (data not shown). This result suggests that the investigated catalytic subunit of type 1 protein phosphatase is at least not the signaling component acted on by NO.

7 Inhibition of BL-specific stomatal opening by NO 721 Action mechanisms of NO We previously indicated that H 2 O 2 produced by ABA inhibits the blue light response of stomata via the suppression of H þ -ATPase phosphorylation (Zhang et al. 2004). In the present study, we determined the effects of ABA and H 2 O 2 on the phosphorylation of both Vfphots and H þ -ATPase in response to blue light. We showed that both ABA and H 2 O 2 only slightly affected the autophosphorylation of Vfphots, but severely inhibited the phosphorylation of H þ -ATPase (Fig. 6). Therefore, the effects of NO on the phosphorylation of Vfphots and the H þ -ATPase were similar to those of H 2 O 2 and ABA. The simplest interpretation of the results could be that NO is produced secondarily by H 2 O 2 that is generated by ABA in guard cells, as has been suggested in the event of stomatal closure of the epidermal peels (L8 et al. 2005, Bright et al. 2006), and that NO is the substance that transmits the signal from H 2 O 2 to the target. If so, we can expect that c-ptio, an NO scavenger, restores the blue light responses that ABA and H 2 O 2 inhibit. However, c-ptio restored the ABA-induced inhibition of stomatal opening by half and did not restore the H 2 O 2 -induced inhibition of stomatal opening. In accord with this result, c-ptio partially restored the ABA-induced inhibition of blue light-dependent H þ pumping and H þ -ATPase phosphorylation, and did not restore the H 2 O 2 -induced inhibition of these two responses (Fig. 7). Since NO is demonstrated to be produced by the action of H 2 O 2, the results suggest that both H 2 O 2 and NO inhibit H þ -ATPase phosphorylation, although the separate production of H 2 O 2 and NO could not be excluded. From these results, we conclude that ABA produces H 2 O 2 and NO, and that NO and H 2 O 2 individually inhibit blue light signaling. It is intriguing that the two distinct small molecules NO and H 2 O 2 exhibit individual but similar effects on the blue light-induced stomatal opening. It is possible that NO and H 2 O 2 affect the same target in the blue light signaling pathways of guard cells. In agreement with this possibility, both NO and H 2 O 2 stimulate stomatal closure via the elevation of cytosolic Ca 2þ in guard cells, and the elevated cytosolic Ca 2þ inhibits both inward-rectifying K þ channels and the plasma membrane H þ -ATPase, and activates the anion channels (Kinoshita et al. 1995, Hamilton et al. 2000, Pei et al. 2000, Schroeder et al. 2001, Garcia-Mata et al. 2003). A recent study demonstrated that the elevated cytosolic Ca 2þ induced by ABA activates the anion channel via the mediation of the Ca 2þ -dependent protein kinases CPK6 and CPK3 in A. thaliana (Mori et al. 2006). Furthermore, inhibition of the H þ -ATPase by CDPK has been reported in the membrane proteins of beet roots (Lino et al. 1998). Further investigation will be needed to clarify the target of NO in guard cell signaling. ABA enhances the tolerance to drought stress It is important for plants to adjust their stomata quickly to appropriate pore sizes so as to prevent water loss under a variety of environments (Assmann and Shimazaki 1999, Roelfsema and Hedrich 2005, Vavasseur and Raghavendra 2005). ABA has been demonstrated to drive stomatal closure via the activation of anion channels that result in membrane depolarization (Schroeder et al. 2001), with a subsequent activation of depolarization of activated outward-rectifying K þ channels. However, if ABA does not inhibit the plasma membrane H þ -ATPase that is activated by blue light, the membrane potential is maintained in a more hyperpolarized state, which antagonizes stomatal closure. Thus, it is important that ABA inhibits the plasma membrane H þ -ATPase, with simultaneous activation of closure systems. In this study, we demonstrated that ABA inhibits blue light-specific stomatal opening through the action of both NO and H 2 O 2 in guard cells. NO and H 2 O 2 are probably synthesized by nitrate reductase and generated by NADPH oxidase in the plasma membrane (Pei et al. 2000, Desikan et al. 2002, Kwak et al. 2003, Bright et al. 2006), respectively, in guard cells. Such distinct mechanisms of production of NO and H 2 O 2 may guarantee stomatal closure and enhance the plant s tolerance to water stress under rapidly changing environments. Materials and Methods Plant materials and isolation of guard cell protoplasts Plants of V. faba (cv. Ryosai Issun) were cultured hydroponically in a greenhouse as described previously (Shimazaki et al. 1992). Guard cell protoplasts were isolated enzymatically from the lower epidermis of 4- to 8-week-old leaves according to a previously described method (Kinoshita and Shimazaki 1999). Isolated guard cell protoplasts were stored in 0.4 M mannitol and 1 mm CaCl 2 on ice in the dark until use. The amount of protein was determined by the method of Bradford (1976) using bovine serum albumin as a standard. Stomatal bioassays Stomatal aperture was determined as described previously (Kinoshita and Shimazaki 1997) with some modifications. The epidermal strips were peeled off from the abaxial side of the leaf under dim light using forceps, and were pre-incubated in a Petri dish containing 10 mm MES-NaOH (ph 6.1), 5 mm KCl and 0.1 mm CaCl 2 for 2 h in the dark. Then, the epidermal strips were illuminated with red light (150 mmol m 2 s 1 ) or with blue light (15 mmol m 2 s 1 ) superimposed on the red light in the presence or absence of treatment reagents for another 2 h. The strips were subsequently examined under a microscope (Eclipse TS100; Nikon, Tokyo, Japan) to determine the aperture of the stomata. Measurements of blue light- and fusicoccin-dependent H þ pumping Blue light-dependent H þ pumping by guard cell protoplasts was determined with a glass ph electrode connected to a ph meter (Shimazaki et al. 1986, Shimazaki et al. 1992). The reaction mixture (1.0 ml) consisted of mm MES-NaOH ph 6.0, 1 mm

8 722 Inhibition of BL-specific stomatal opening by NO CaCl 2, 0.4 M mannitol, 10 mm KCl and guard cell protoplasts (50 mg of protein). Blue light (100 mmol m 2 s 1 ) was applied as a 30 s pulse 40 min after the onset of background red light (600 mmol m 2 s 1 ). Red light was obtained from a tungsten lamp (Philips EXR 300 W) by passing the light through a red glass filter (Corning 2-61). Blue light was obtained from a tungsten lamp (Sylvania EXR 150 W) by passing the light through a blue glass filter (Corning 5-60). Photon flux density was measured with a quantum meter (model 185a, Li-Cor, Lincoln, NE, USA). Fusicoccin-dependent H þ pumping by guard cell protoplasts was determined in the same reaction mixture under irradiation with red light (600 mmol m 2 s 1 ). To examine the effects of NO on blue light-dependent H þ pumping, SNP (an NO donor) was added to guard cell protoplast suspensions at the indicated times before the application of the blue light pulse. c-ptio was added to guard cell protoplast suspensions at the onset of background red light. All of the measurements were performed at 248C. Immunoblot detection The plasma membrane H þ -ATPase was detected immunologically with rabbit antisera raised against H þ -ATPase according to the methods described previously (Kinoshita and Shimazaki 1999, Zhang et al. 2004). Protein blot analysis The binding of a protein to H þ -ATPase was analyzed according to a previously described method (Kinoshita and Shimazaki 1999, Kinoshita et al. 2003). Determination of levels of phosphorylation and amounts of Vfphots and H þ -ATPase The phosphorylation levels of Vfphots and H þ -ATPase were determined using guard cell protoplasts that had been labeled with 32 P, as described previously (Kinoshita et al. 2003). The amounts of Vfphots and H þ -ATPase were determined immunologically. For detection of Vfphots, the reaction was terminated by adding trichloroacetic acid. For detection of H þ -ATPase, the reaction was terminated by adding an equal volume of the medium containing 100 mm MOPS-KOH (ph 7.5), 5 mm EDTA, 200 mm NaCl, 1 mm phenylmethylsulfonyl fluoride (PMSF), 20 mm leupeptin, 4 mm dithiothreitol (DTT), 20 mm NaF, 1 mm ammonium molybdate, 100 nm calyculin A and 2% (w/v) Triton X-100. Immunoprecipitation was performed using the anti-h þ -ATPase antibodies conjugated to protein A agarose with dimethyl pimelimidate dihydrochloride (DMP). The process was described in detail previously (Takemiya et al. 2006). Determination of H þ pumping in microsomal membranes A membrane fraction that contained vanadate-sensitive H þ transport activity was obtained according to a previously described method (Goh et al. 1996). Acknowledgments This work was supported by Grants-in-aid for Scientific Research (Nos and to K.S.) from the Ministry of Education, Science, Sports, and Culture, and by Grant-in-aid (No to X.Z.) from the National Natural Science Foundation of China. References Assmann, S.M. and Shimazaki, K. (1999) The multisensory guard cell, stomatal responses to blue light and abscisic acid. Plant Physiol. 119: Assmann, S.M., Simoncini, L. and Schroeder, J.I. (1985) Blue light activates electrogenic ion pumping in guard cell protoplasts of Vicia faba. Nature 318: Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72: Briggs, W.R., Beck, C.F., Cashmore, A.R., Christie, J.M., Hughes, J., et al. (2001) The phototropin family of photoreceptors. Plant Cel 13: Briggs, W.R. and Christie, J.M. (2002) Phototropins 1 and 2: versatile plant blue-light receptors. Trends Plant Sci. 7: Bright, J., Desikan, R., Hancock, J.T., Weir, I. and Neill, S.J. (2006) ABAinduced NO generation and stomatal closure in Arabidopsis are dependent on H 2 O 2 synthesis. Plant J. 45: Christie, J.M., Reymond, P., Powell, G.K., Bernasconi, P., Raibekas, A.A., Liscum, E. and Briggs, W.R. (1998) Arabidopsis NPH1: a flavoprotein with the properties of a photoreceptor for phototropism. Science 282: Delledonne, M., Xia, Y., Dixon, R.A. and Lamb, C. (1998) Nitric oxide functions as a signal in plant disease resistance. Nature 394: Desikan, R., Griffiths, R., Hancock, J.T. and Neill, S.J. (2002) A new role for an old enzyme: nitrate reductase-mediated nitric oxide generation is required for abscisic acid-induced stomatal closure in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 99: Folta, K.M., Lieg, E.J., Durham, T. and Spalding, E.P. (2003) Primary inhibition of hypocotyl growth and phototropism depend differently on phototropin-mediated increases in cytoplasmic calcium induced by blue light. Plant Physiol. 133: Furchgott, R.F. (1995) Special topic: nitric oxide. Annu. Rev. Physiol. 57: Garcia-Mata, C., Gay, R., Sokolovski, S., Hills, A., Lamattina, L. and Blatt, M.R. (2003) Nitric oxide regulates K þ and Cl channels in guard cells through a subset of abscisic acid-evoked signaling pathways. Proc. Natl Acad. Sci. USA 100: Garcia-Mata, C. and Lamattina, L. (2001) Nitric oxide induces stomatal closure and enhances the adaptive plant responses against drought stress. Plant Physiol. 126: Garcia-Mata, C. and Lamattina, L. (2002) Nitric oxide and abscisic acid cross talk in guard cells. Plant Physiol. 128: Garcia-Mata, C. and Lamattina, L. (2003) Abscisic acid, nitric oxide and stomatal closure. Is nitrate reductase one of the missing links? Trends Plant Sci. 8: Goh, C.H., Kinoshita, T., Oku, T. and Shimazaki, K. (1996) Inhibition of blue light-dependent H þ pumping by abscisic acid in Vicia guard-cell protoplasts. Plant Physiol. 111: Gould, K.S., Lamotte, O., Klinguer, A., Pugin, A. and Wendehenne, D. (2003) Nitric oxide production in tobacco leaf cells: a generalized stress response? Plant Cell Environ. 26: Hamilton, D.W.A., Hill, A., Kohler, B. and Blatt, M.R. (2000) Ca 2þ channels at the plasma membrane of stomatal guard cells are activated by hyperpolarization and abscisic acid. Proc. Natl Acad. Sci. USA 97: Huala, E., Oeller, P.W., Liscum, E., Han, I.S., Larsen, E. and Briggs, W.R. (1997) Arabidopsis NPH1: a protein kinase with a putative redox-sensing domain. Science 278: Inada, S., Ohgishi, M., Mayama, T., Okada, K. and Sakai, T. (2004) RPT2 is a signal transducer involved in phototropic response and stomatal opening by association with phototropin1 in Arabidopsis thaliana. Plant Cell 16: Inoue, S., Kinoshita, T. and Shimazaki, K. (2005) Possible involvement of phototropins in leaf movement of kidney bean in response to blue light. Plant Physiol. 138: Kagawa, T., Sakai, T., Suetsugu, N., Oikawa, K., Ishiguro, S., Kato, T., Tabata, S., Okada, K. and Wada, M. (2001) Arabidopsis NPL1: a

9 Inhibition of BL-specific stomatal opening by NO 723 phototropin homolog controlling the chloroplast high-light avoidance response. Science 291: Kinoshita, T., Doi, M., Suetsugu, N., Kagawa, T., Wada, M. and Shimazaki, K. (2001) phot1 and phot2 mediate blue light regulation of stomatal opening. Nature 414: Kinoshita, T., Emi, T., Tominaga, M., Sakamoto, K., Shigenaga, A., Doi, M. and Shimazaki, K. (2003) Blue-light- and phosphorylationdependent binding of a protein to phototropins in stomatal guard cells of broad bean. Plant Physiol. 133: Kinoshita, T., Nishimura, M. and Shimazaki, K. (1995) Cytosolic concentration of Ca 2þ regulates the plasma membrane H þ -ATPase in guard cells of fava bean. Plant Cell 7: Kinoshita, T. and Shimazaki, K. (1997) Involvement of calyculin A- and okadaic acid-sensitive protein phosphatase in the blue light response of stomatal guard cells. Plant Cell Physiol. 38: Kinoshita, T. and Shimazaki, K. (1999) Blue light activates the plasma membrane H þ -ATPase by phosphorylation of the C-terminus in stomatal guard cells. EMBO J. 18: Kinoshita, T. and Shimazaki, K. (2001) Analysis of the phosphorylation level in guard-cell plasma membrane H þ -ATPase in response to fusicoccin. Plant Cell Physiol. 42: Kinoshita, T. and Shimazaki, K. (2002) Biochemical evidence for the requirement of protein binding in activation of the guard-cell plasma membrane H þ -ATPase by blue light. Plant Cell Physiol. 43: Kwak, J.M., Mori, I.C., Pei, Z.M., Leonhardt, N., Torres, M.A., Dangl, J.L., Bloom, R.E., Bodde, S., Jones, J.D. and Schroeder, J.I. (2003) NADPH oxidase AtbohD and AtbohF genes function in ROSdependent ABA signaling in Arabidopsis. EMBO J. 22: Lino, B., Baizbal-Aguirre, V.M. and de la Vara, L.E.G. (1998) The plasmamembrane H þ -ATPase from beet root is inhibited by a calciumdependent phosphorylation. Planta 204: L8, D., Zhang, X., Jiang, J., An, G.Y., Zhang, L.R. and Song, C.P. (2005) NO may function in the downstream of H 2 O 2 in ABA-induced stomatal closure in Vicia faba L. J. Plant Physiol. Mol. Biol. 31: Mori, I.C., Murata, Y., Yang, Y., Munemasa, S., Wang, Y.F., et al. (2006) CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and Ca 2þ - permeable channels and stomatal closure. PLoS Biol. 4: Neill, S.J., Desikan, R., Clarke, A. and Hancock, J.T. (2002a) Nitric oxide is a novel component of abscisic acid signaling in stomatal guard cell. Plant Physiol. 128: Neill, S.J., Desikan, R., Clarke, A., Hurst, R.D. and Hancock, J.T. (2002b) Hydrogen peroxide and nitric oxide as signalling molecules in plants. J. Exp. Bot. 53: Pei, Z.M., Murata, Y., Benning, G., Thomine, S., Klusener, B., Allen, G.J., Grill, E. and Schroeder, J.L. (2000) Calcium channels activated by hydrogen peroxide mediate abscisic acid signaling in guard cells. Nature 406: Roelfsema, M.R.G. and Hedrich, R. (2005) In the light of stomatal opening: new insights into the Watergate. New Phytol. 167: Roelfsema, M.R.G., Steinmeyer, R., Staal, M. and Hedrich, R. (2001) Single guard cell recordings in intact plants: light-induced hyperpolarization of the plasma membrane. Plant J. 26: Sakai, T., Kagawa, T., Kasahara, M., Swartz, T.E., Christie, J.M., Briggs, W.R., Wada, M. and Okada, K. (2001) Arabidospsis nph1 and npl1: blue light receptors that mediate both phototropism and chloroplast relocation. Proc. Natl Acad. Sci. USA 98: Sakamoto, K. and Briggs, W.R. (2002) Cellular and subcellular location of phototropin1. Plant Cell 14: Salomon, M., Christie, J.M., Knieb, E., Lempert, U. and Briggs, W.R. (2000) Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin. Biochemistry 39: Schroeder, J.I., Allen, G.J., Hugouvieux, V., Kwak, J.M. and Waner, D. (2001) Guard cell signal transduction. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52: Shimazaki, K., Doi, M., Assmann, S.M. and Kinoshita, T. (2007) Light regulation of stomatal movement. Ann. Rev. Plant Biol. 58: Shimazaki, K., Goh, C.H. and Kinoshita, T. (1999) Involvement of intracellular Ca 2þ in blue light-dependent proton pumping in guard cell protoplasts from Vicia faba. Physiol. Plant 105: Shimazaki, K., Iino, M. and Zeiger, E. (1986) Blue light-dependent proton extrusion by guard-cell protoplasts of Vicia faba. Nature 319: Shimazaki, K., Kinoshita, T. and Nishimura, M. (1992) Involvement of calmodulin and camodulin-dependent myosin light chain kinase in blue light-dependent H þ pumping by guard cell protoplasts from Vicia faba L. Plant Physiol. 99: Sokolovski, S. and Blatt, M.R. (2004) Nitric oxide block of outwardrectifying K þ channels indicates direct control by protein nitrosylation in guard cells. Plant Physiol. 136: Sokolovski, S., Hills, A., Gay, R., Garcia-Mata, C., Lamattina, L. and Blatt, M.R. (2005) Protein phosphorylation is a prerequisite for intracellular Ca 2þ release and ion channel control by nitric oxide and abscisic acid in guard cells. Plant J. 43: Svennelid, F., Olsson, A., Piotrowski, M., Rosenquist, M., Ottman, C., Larsson, C., Oecking, C. and Sommarin, M. (1999) Phosphorylation of Thr-948 at the C terminus of the plasma membrane H þ -ATPase creates a binding site for the regulatory protein. Plant Cell 11: Takemiya, A., Kinoshita, T., Asanuma, M. and Shimazaki, K. (2006) Protein phosphatase 1 positively regulates stomatal opening to blue light in Vicia faba. Proc. Natl Acad. Sci. USA 103: Taylor, A.R. and Assmann, S.M. (2001) Apparent absence of a redox requirement for blue light activation of pump current in broad bean guard cells. Plant Physiol. 125: Vavasseur, A. and Raghavendra, A.S. (2005) Guard cell metabolism and CO 2 sensing. New Phytol. 165: Zhang, X., Wang, H.B., Takemiya, A., Song, C.P., Kinoshita, T. and Shimazaki, K. (2004) Inhibition of blue light-dependent H þ pumping by abscisic acid through hydrogen peroxide-induced dephosphorylation of the plasma membrane H þ -ATPase in guard cell protoplasts. Plant Physiol. 136: Zhang, X., Zhang, L., Dong, F.C., Gao, J.F., Galbraith, D.W. and Song, C.P. (2001) Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba. Plant Physiol. 126: (Received February 5, 2007; Accepted March 20, 2007)